6th World Melioidosis Congress

Identifying and unravelling the known unknowns in melioidosis epidemiology. Bart Currie. Melioidosis is a major cause of disease in Thailand, Malaysia, Singapore and Northern Australia. In Cambodia there are significant differences within the country e.g. not many cases in Phnom Penh, but a lot in rural locations such as Siem Reap. Global spread with introduction into SE Asia, is believed to have occurred during the last Ice Age. There are unexplained locations such as Aruba, East and West Africa, Madagascar and desert locations such as Arizona. Dissemination has been proposed via animals or plants and soil, but long range transfer via human travel or by birds has not yet been demonstrated. Environmental determinants have been identified e.g. rice paddies, exotic animals, bore water supplies, sports fields and savannah grasses and landscape changes are not been documented and used to develop predictive maps. Genome analysis has shown evidence of horizontal gene transfer from other bacteria including other Burkholderia species that co-habit with Burkholderia pseudomallei, and identified over 100 gene islands. But it still remains to be seen whether B. pseudomallei is a true biothreat agent or an environmental opportunist. In recent reviews of NT culture positive cases, 14% had fatal infections but none of these were previously healthy. It is still not clear whether there is a subset of really nasty strains among the clinical variants such as those responsible for neurological infection. The mode of infection is believed to be mainly inoculation, though severe weather is thought to cause a shift in favour of inhalation. The global extent of environmental B. pseudomallei is not known, nor its means of long distance spread, nor the environmental drivers of bacterial load.

Development of Ceftazidime resistance by Burkholderia pseudomallei following human infection associated with large scale gene deletion. Narisara Chantratita. B. pseudomallei was isolated from a patient with multiple splenic abscesses who failure therapy. The isolate had a Ceftazidime resistant phenotype (MIC > 256 ug/mL), was nutritionally variant and produced filamentous forms. It only grew on Ashdown’s agar, and not on other solid media. MLST genotyping indicated that it probably developed from the original B. pseudomallei isolate. A comparative genomic hybridisation array showed a 71kb deletion of 49 genes on chromosome 2 associated with penicillin binding protein.

Glibenclamide is anti-inflammatory and associated with reduced mortality in patients with septic melioidosis. G Koh

Glibenclamide is anti-inflammatory and associated with reduced mortality in patients with septic melioidosis. Gavin Koh. In NE Thailand the majority of patients with septicaemic melioidosis have diabetes, but interestingly there is a reduced risk of death, contrary to expectations based on the diabetic mouse model. Careful epidemiological analysis showed that the survival benefit was restricted to patients receiving glibenclamide who had a 50% reduction in risk. The precise mechanism of action is unclear but there is no direct inhibition of B. pseudomallei growth. However glibenclamide was shown to reduce inflammasome assembly-related gene expression in polymorphonuclear leukocytes.

Melioidosis in Cambodia: clinical and epidemiological data in 58 patients. Erika Vleighe. The first work on melioidosis in Cambodia was reported in 2008 by Wuthiekanun and colleagues who studied the epidemiology of an apparently emerging infection. There were then two documented clinical cases. Prospective studies on melioidosis have identified 58 cases since then, mainly from southern lowlands and particularly in diabetic farmers. 11.5% blood culture isolates in the Prince Sihanouk Hospital grew B. pseudomallei. A high proportion of septicaemic cases developed septic shock.

Reservoir for melioidosis in Townsville city. Anthony Baker. Melioidosis cases in Townsville cluster around Castle Hill. An environmental sampling method was developed but due to Taq polymerase inhibition, direct NAA was abandoned in favour of an initial culture step followed by PCR. Systematic sampling was planned in transects, taking 50g soil at 30cm depth. A maximum of 114 CFU/mL B pseudomallei was recorded. Run off at W and SW from Castle Hill gave 14/16 PCR positive locations. A totl of 10 MLST types were found, with multiple sequence types in single water samples.

Utility of culture in the molecular era for the diagnosis of melioidosis. Vanaporn Wuthiekanun. In Thailand there has been an increase in melioidosis incidence from 9 to 20 per 100,000 population. Direct immunofluorescence is not commercially available. LAMP and conventional PCR methods based e.g. on the TTSS cluster have low sensitivity due to the low number of bacterial cells present. Therefore we need to reconsider the role of culture and confirmation of identity by phenotypic methods such as API 20NE. From 1997-2006 2243 culture positive infections were followed. Blood cultures were collected on admission and after 10 days. 956 patients died (43%). The bacterial count was greatest is pus samples and lowest in blood cultures. The presence of B. pseudomallei in urine was associated with a poor prognosis, but only 24% patients with a positive MSU had urinary symptoms. A positive throat swab was 100% specific, but only 36% sensitive.Only 2 patients in more than 2000 had a Ceftazidime resistant strain on admission and in vitro antibiotic resistance developed in only 25.

Defining the true accuracy of diagnostic tests for the diagnosis of melioidosis using Bayesian latent class models. Direk Limmathurotsakul. The problem with diagnostic tests for melioidosis is that the gold standard is imperfect. The true sensitivity of tests for melioidosis is unknown, Hypothetical assessment suggests an apparently poor specificity and thus severe bias. Bayesian analysis has been applied to diagnostic tests for several decades. Given three tests (culture, IHA, IgM ELISA) applied to a single patient population, a mathematical model was developed, using Markov Chain Monte Carlo and a random walk with WinBUGS. A Bayesian latent class model predicts 60% sensitivity for blood culture and 70% for IHA. ELISA has a lower predicted sensitivity but much higher predicted specificity. Is this hypothetical or actual? Culture should probably not be used as a gold standard test.

Molecular basis of antibiotic resistance mechanisms in Burkholderia pseudomallei: lessons for rational melioidosis therapy. Herbert Schweizer. Effective antibiotic therapy needs to take account of intrinsic and acquired mechanisms of antibiotic resistance. A series of tools compliant with Select Agent regulations have been designed. Two main mechanisms of resistance operate: PenA, a class A beta-lactamase, and BpeEF-OprC, an efflux pump belonging to the resistance-nodulation-cell division superfamily (RND). PenA acts on beta-lactams including Ceftazidime. Some efflux pump mutants have reduced susceptibility to a specific therapeutic agent on exposure to subinhibitory concentrations of Trimethoprim or Doxycycline, so that resistance becomes dependent on the other antibiotic. Inhibitory combinations should be discouraged. Meropenem is preferable to Imipenem.

Burkholderia mallei and Burkholderia pseudomallei genomic analysis, evolution and insights into pathogen virulence. Paul Keim. The genome of B. pseudomallei is more than twice the size of Staphylococcus aureus, has two chromosomes and a high GC ration. It is difficult to sequence. The Sanger Centre’s work on B. pseudomallei K96243 provided a critical resource. Gene islands are easy to locate from the GC ratio. If a bacterial genome is clonally propagated, genomic diversity is driven by mutation. B. pseudomallei achieves diversity by lateral gene transfer including from other bacterial species e.g. Yersinia-like chemotaxis factor genes.10-15% difference between genomes corresponds to adaptation to different environments. In B. pseudomallei the extent of recombination contributing accessory genes beyond the core genome is extreme, and differs between Australian and SE Asian strains. A novel system of analysis has been developed around obligatory orthologous SNPs.

B. mallei, on the other hand, has fewer additional genes on new strain genomes. This is a closed and cloistered genome. It has a 0.84 consistency index, suggesting clonal propagation. However, there has is still some genetic variation due to an explosion of IS elements consistent with specialisation. Genome decay relates to adaptation or inhibition. B. pseudomallei, by comparison, has a promiscuous genome.

One instance of B. pseudomallei evolution has been noted involving a eight year series in an animal model. Involving two missing regions, and a 200kb deletion. This may be similar to the natural process in B. mallei. There will be intra-host neutral variation. Hypermutable regions are subject to genetic decay. Selected variation is likely to be a very rare event. E.coli O157:H7 has a highly clonal, specialised genome with little evidence of horizontal gene transfer. The problem with next generation sequencing that relies on autoassembly is in seeing something new.

A genomic survey of positive selection on Burkholderia pseudomallei provides insights into the evolution of accidental virulence. Tannistha Nandi. B. pseudomallei shows evidence of a complex co-evolutionary process. Adaptation has resulted from a range of selection pressures, possibly contributing to the emergence of virulence. B. pseudomallei genomes were sequentially analysed: SNPs, indels (minor fractions) and SNP microgenome variations corresponding to geographic location. A positive selection process led to selection of a range of adherence, membrane functions and stress response. A proportion of the core genome is subject to functional selection, leading to accidental pathogenesis.

Association of soil properties, landscape position and causative agent with melioidosis case distribution in Townsville, North Queensland. Maree Corkeron. Spatial clusters of melioidosis occur around the Ross River. Statistical analysis of clusters and their relationship to five key soil landscape types including Piedmont slopes, undulating uplands & alluvial plane deposits. Analysis included clay abundance, mineral content, pH and topography. The landscape model shows depressions, flats, slopes and crests. Cases were located more with flats, than depressions, slopes or crests, contrary to expectation based on depth and waterlogging.

Effects of landscape change ecology upon Burkholderia pseudomallei in the Top End of Australia. Mirjam Kaestli. B. pseudomallei has been associated with disturbed soils in gardens. Organic and NPK fertilisers are both associated with increased B. pseudomallei counts. Analyses at test sites have included soil moisture, clay content, urea, soil pH and organic fertiliser. An association has been made with invasive mission grass. Hotspots have been determined that correlate with introduced Tully, Paspalum and Mission grass varieties. It remains to be seen whether bacteria and grasses prefer the same environment or have a specific association. However, fluorescent in situ hybridisation shows B. pseudomallei outside the stomata and in the root hairs of grasses in vitro. Wild rice, however, was not affected.